Maintaining the integrity of the genome is crucial in ensuring cellular homeostasis. One of the most cytotoxic forms of genomic damage is the DNA double strand break (DSB). DSBs can arise as a result of exposure to DNA damaging agents or during normal DNA metabolic processes. In addition, programmed DSBs are generated via highly regulated processes such as the lymphoid specific DNA rearrangement, V(D)J recombination. If unrepaired or misrepaired, DSBs can lead to increased genome instability and accumulation of aberrant chromosomal rearrangements which can result in severely detrimental outcomes for cells and organisms. The non-homologous end joining (NHEJ) pathway is one of the major DNA DSB repair pathways in mammalian cells and is required for general DSB repair as well as V(D)J recombination. Many chromosomal DSBs, including those generated during V(D)J recombination, have end structures that require modification prior to joining. Thus, one important event that occurs during NHEJ is the processing of the DNA ends to prepare them for ligation. The Artemis DNA nuclease, in complex with the DNA-PKcs protein kinase, plays a critical role as an endonuclease and can cleave DNA substrates at single to double strand transitions, such as flaps, overhangs, loops and hairpins. Another nuclease that has important functions during DSB repair is Mre11 which functions in the context of the Mre11/Rad50/Nbs1 (MRN) complex. Several lines of evidence suggest that Artemis and Mre11 may function in concert during the repair of DNA DSBs. However, the precise roles and functional interactions between Artemis and MRN have not been elucidated. Mutations in either Artemis or Mre11 result in human immunodeficiencies associated with genome instability, and in some cases, cancer predisposition. Thus, the major goals of the current proposal are to gain a better understanding of molecular and functional interactions between Artemis and Mre11 that are involved in DNA end processing in the context of lymphocyte development and general DSB repair. To this end, three specific aims are proposed. The goals of Aim 1 are to elucidate the functional interactions between the Artemis and Mre11 DNA nucleases during V(D)J recombination using novel mouse models harboring conditional, null, and hypomorphic knock-in alleles. Aim 2 Is to examine the roles of Mre11 in tumorigenesis in the context of defective V(D)J recombination. In Aim 3, we propose experiments to elucidate the functional interactions between Artemis and Mre11 during general DNA DSB repair. Together, these studies will provide important insights into the molecular events that ensure the efficient joining of broken chromosomal ends, a vital process required for maintenance of genome stability and immune system development. Furthermore, the proposed studies will provide a more in-depth understanding of the mechanisms underlying the generation of oncogenic events that lead to tumorigenesis. PUBLIC HEALTH RELEVANCE: This proposal will examine the molecular mechanisms underlying the repair of broken chromosomal ends and determine the consequences of aberrant DNA end processing on immune system development and lymphoid malignancies. Together, these studies will not only provide important insights into the basic processes required for maintenance of genome stability, but also lead to a better understanding of the causes of human immunodeficiency and cancer predisposition syndromes.